WO2001039594A2 - Cryoconservation of de cellules dendritiques chargees d'antigenes et leurs precurseurs dans des milieux sans serum - Google Patents

Cryoconservation of de cellules dendritiques chargees d'antigenes et leurs precurseurs dans des milieux sans serum Download PDF

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WO2001039594A2
WO2001039594A2 PCT/US2000/032722 US0032722W WO0139594A2 WO 2001039594 A2 WO2001039594 A2 WO 2001039594A2 US 0032722 W US0032722 W US 0032722W WO 0139594 A2 WO0139594 A2 WO 0139594A2
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precursors
apc
cryopreservation
cells
serum
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PCT/US2000/032722
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WO2001039594A3 (fr
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Tatsue Monji
Madhusudan V. Peshwa
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Dendreon Corporation
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Publication of WO2001039594A3 publication Critical patent/WO2001039594A3/fr

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    • A01N1/0221—
    • A01N1/02—

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  • the present invention relates to compositions and methods relating to cryopreservation of dendritic cell (DC) precursors, and DC products.
  • DC dendritic cell
  • DC are effective antigen presenting cells (APC) . They are potent allostimulators and the only APC capable of priming naive helper and cytotoxic T lymphocytes. In T cell-mediated cancer immunotherapy, induction of tumor-specific immune response depends on effective antigen (Ag) presentation. Because of their unique ability to prime naive T lymphocytes, DC have been found to be promising in the treatment of various cancers such as B cell lymphoma, melanoma, prostate cancer, and breast cancer. In a general exemplary treatment protocol, DC precursors collected from either the bone marrow or peripheral blood of a patient are cultured ex vivo in the presence of tumor Ag .
  • APC effective antigen presenting cells
  • DC precursors process tumor Ag and differentiate into mature, Ag-loaded DC.
  • the Ag-loaded DC are then reinfused into patients, where the APC induce immune responses directed to cancer Ag.
  • DC precursors are cultured immediately after collection from the patient, e.g. a cancer patient, and Ag-loaded mature DC reinfused into the patient as soon as they are harvested.
  • time constraints not only limit the potential therapeutic uses for DC, but also necessitate leukapheresis products from a patient at multiple time points during the course of immunotherapy.
  • Ag-loaded DC have already processed the Ag and have the ability to present Ag to the immune cells and can therefore quickly generate Ag-specific immune responses. Cryopreservation of Ag-loaded mature DC in a functionally potent state has not been demonstrated to date.
  • Standard freezing medium for cultured cells includes about 5-10% dimethyl sulfoxide (D SO) or glycerol and 10-50% serum (Voetman AA, et al . , Blood 63(l):234-7, 1984; Utsugi T et al., Biotherapy 5(4):301-8, 1992).
  • D SO dimethyl sulfoxide
  • glycerol glycerol
  • serum glycerol
  • Some procedures utilize controlled-rate freezing while others include an insulated container in which vials of cells m freezing medium are placed in a freezer (i.e., at -70 to -80°C) .
  • the present invention provides methods and compositions for cryopreservation of APC precursors and APC.
  • a cryopreservation composition comprising APC precursors or APC is prepared by obtaining a blood sample from a subject, treating the blood sample in a manner effective to obtain a population of APC precursors or APC and suspending the APC precursors or APC in serum-free cryopreservation medium containing DMSO, such that APC precursors or APC frozen to a temperature of -70°C or lower for at least 24 hours in the cryopreservation composition maintain viability with a recovery of at least 70%, upon thawing the cells.
  • the cryopreservation method includes bouyant density cell separation to obtain from a blood sample, a cell fraction containing peripheral blood lymphocytes and DC precursors. In some cases, this fraction is cultured in a serum- free or protein-free medium for a period sufficient to produce morphological, phenotypic and functional changes in DC precursors to cells having the morphology, phenotype and function of DC.
  • the APC precursors are DC precursors which maintain the phenotype and function of DC precursors upon thawing.
  • the APC are Ag-loaded DC, which maintain the phenotype and Ag-specific function of Ag-loaded DC upon thawing the cells.
  • the invention further provides a serum-free cryoprotective medium containing from about 5 to 20% DMSO (volume percent) , for cryopreservation of APC precursors and APC, such that APC precursors or APC frozen to a temperature of -70°C or lower in the cryopreservation medium for at least 24 hours maintain a viability and recovery of at least 70% upon thawing the cells.
  • the cryoprotective medium is protein-free and may contain from about 1 to 30% human serum albumin (HSA) .
  • the cryoprotective medium contains a commercially available serum-free medium, exemplified by AIM-V, XVIVO 10, XVIVO 15, XVIVO 20 and StemPro.
  • the invention provides a method for cryopreserving and cryoprotecting APC precursors and APC by obtaining a blood sample from a subject; treating the blood sample in a manner effective to obtain a population of APC precursors or APC; and suspending the APC precursors or APC in a serum-free cryopreservation medium containing DMSO, such that APC precursors or APC frozen to a temperature of -70°C or lower for at least 24 hours in the cryopreservation medium maintain a viability and recovery of at least 70% upon freezing and thawing.
  • the APC precursors may be DC precursors which maintain the phenotype and function of DC precursors upon thawing and the APC may be mature Ag-loaded DC, which maintain the phenotype and Ag-specific function of DC upon thawing the cells.
  • Figures 1A-Q depict the results of 3-color FACS analysis for expression of the DC-associated markers: CDllc (Fig. IB, 1G, 1M) , CD40 (Fig. 1C, 1H, IN), CD54 (Fig. ID, 1J, IP), CD86 (Fig. IE, IK, 1Q) , and HLA-DR on the lineage-negative population, where the expression on fresh DC (Fig. 1A-E ), DC generated from cryopreserved precursors (Fig. 1F-K) and DC cryopreserved as DC products (Fig. 1L-Q) , was evaluated.
  • Figures 2A-C depict the results of allogeneic and autologous T cell proliferation assays.
  • Fig. 2A presents the results of an analysis of the stimulation of allogeneic T cells
  • Figures 3A-D and 3E-H depict the results of FACS analysis for expression of CD54 on DC generated from cryopreserved precursors (Fig. 3A-D) and DC cryopreserved as DC products (Fig. 3E-H) following 0 (3A, 3E) , 4 (3B, 3F) , 8 (3C, 3G) and 24 (3D, 3H) hours in lactated Ringer's solution at 4°C.
  • Figures 4A and 4B depict the results of an analysis of the stimulation of allogeneic T cells by DC cryopreserved as DC precursors, then cultured in the presence of recombinant PA2024 (Fig.
  • DC precursors are peripheral blood cells which can mature into DC under suitable conditions.
  • DC precursors may comprise a mixture of peripheral blood cells and typically have a non-dendritic morphology and are not competent to elicit a primary immune response as APC.
  • Dendritic cells or “DC” are mature DC, which typically have a DC morphology, that is, they are large veiled cells which extend dendrites when cultured ex vivo . When pulsed with Ag or peptide, such DC are capable of presenting Ag to naive T cells.
  • DC products refers to Ag-loaded DC generated by ex vivo culture in serum-free or protein-free medium in the presence of antigen, for a time sufficient for the cells to acquire the morphology, phenotype and function of mature DC.
  • mature DC refers to DC generated by ex vivo culture in serum-free medium for a time sufficient for the cells to acquire the phenotype, morphology and function of mature DC.
  • Antigen presenting cell precursors or “APC precursors” are cells that when exposed to differentiation stimulating conditions, e.g., environmental stimuli, cell culture, or cytokines, are capable of becoming mature APC and presenting Ag or peptide to T cells .
  • Antigen presenting cells are cells which when exposed to an Ag or peptide, can activate CD8 + cytotoxic T- lymphocytes (CTL) or CD4 + helper T-lymphocytes in an immune response .
  • CTL cytotoxic T- lymphocytes
  • CD4 + helper T-lymphocytes in an immune response .
  • the term "lin -" refers to a cell population that is negative for cell surface expression of the lineage markers expressed on T cells, B cells, monocytes/macrophages, NK cells, eosinophils, and neutrophils: for example, CD3, CD14, CD16, CD19, CD20, and CD56.
  • allostimulatory means capable of stimulating allogeneic T cells due to differences in MHC molecules expressed on the cell surface.
  • Ag-loaded mature DC refers to peripheral blood mononuclear cell (PBMC) -derived cells enriched for DC precursors and cultured ex vivo in the presence of Ag, e.g., a tumor Ag .
  • PBMC peripheral blood mononuclear cell
  • Such "Ag-loaded mature DC” include DC and various types of PBMC including professional APC, monocytes/macrophages, which are positive for cell surface expression of CD14, and B lymphocytes, which are positive for cell surface expression of CD19.
  • immunogen refers to a substance that is able to stimulate or induce a humoral antibody and/or cell- mediated immune response.
  • Antigen or “Ag” refers to a substance that reacts alone or in the context of MHC molecules with the products of an immune response (e.g., antibodies, T-cell receptors) which have been stimulated by a specific immunogen. Ag therefore include the specific immunogens giving rise to the response (e.g., antigenic peptides, proteins or polysaccharides) as well as the entities containing or expressing the specific immunogens (e.g., viruses, bacteria, etc. ) .
  • Tumor antigens refer to tumor-associated Ag and tumor- specific Ag .
  • tumor Ag include HER-2/neu, prostatic acid phosphate (PAP) and any of a number of proteins expressed on tumor cells.
  • PAP prostatic acid phosphate
  • cryopreserved refers to cells that have been resuspended in a cryomedium, frozen at a temperature of -70°C or lower, and preserved at that temperature for a minimum of 24 hours.
  • DC precursors and DC PBMC were collected from healthy donors by standard leukapheresis and DC precursors isolated by either a one-step or a successive two-step buoyant density centrifugation procedure, as further described below.
  • DC may be generated from such DC precursors by culture ex vivo in serum-free or protein-free medium for 40 hours, in the absence of exogenously added cytokines, as detailed in co-owned
  • the purity of DC in this fraction may be quantified using, for example, flow cytometry (i.e., FACS) analysis, together with functional assays.
  • flow cytometry i.e., FACS
  • the DC population is characterized phenotypically as lineage negative, i . e . , negative for expression of the cell- surface lineage markers CD3, CD14, CD16, CD19, CD20, and CD56; strongly positive for the cell-surface marker CD86 ("CD86- bright"); and positive for cell surface expression of MHC class II molecules such as HLA DR (Lin-/86++/DR+) . They also express DC-associated markers such as CDllc, CD40 and CD54. This Lin-
  • /86++/DR+ population is not detected in freshly isolated PBMC or in cell fractions enriched for DC precursors.
  • DC precursors acquire a mature DC phenotype during the period of ex vivo culture.
  • the cells upregulate expression of CDllc, CD40, CD54, CD86, and MHC class II molecules, as further described in co- owned USSN 09/684,308.
  • the FACS profile of DC generated from fresh precursors, those generated from cryopreserved precursors and those cryopreserved as DC products was compared.
  • the DC culture contains various types of cells such as T lymphocytes, B lymphocytes and monocytes/ macrophages
  • FACS analysis facilitates the analysis of DC lineage cells.
  • DC products were stained with FITC-Lin, PE-CDllc, -CD40, -CD54, or -CD86 and Per-CP-HLA-DR, as detailed in Example 3. The results indicate that: (1) cryopreserved DC precursors maintain the ability of the cells to acquire a mature DC phenotype, and (2) cryopreserved DC products maintain their mature DC phenotype.
  • the acquisition of the mature DC phenotype correlates with the functional maturation of DC, in that DC precursors become APC which are not only allostimulatory but also capable of presenting Ag to autologous T cells.
  • the ability to provide a ready supply of cryopreserved cells of either type, particularly cells which can elicit Ag-specific immune responses represents a significant advantage that can facilitate various therapeutic uses of the cells.
  • a large scale culture of DC precursor cells or Ag-loaded DC may be cryopreserved in aliquots of the appropriate size for individual doses of cells for use in a particular immunotherapeutic protocol .
  • cryopreservation protocols have been reported in the literature for primary cells and cultured cell lines. Examples include: the cryopreservation of enucleated human neutrophils (PMN cytoplasts) in a medium containing 10% (v/v) fetal calf serum and 10% (v/v) DMSO, and stored at -70 °C, resulting in a recovery of 75% (Voetman AA, et al .
  • the present invention is directed to the specific effects of cryopreservation in liquid nitrogen on DC precursors collected from peripheral blood, as well as on mature Ag-loaded DC generated from the DC precursors, with an emphasis on serum-free and protein-free cryomedium and large scale cryopreservation.
  • the effects of cryopreservation on DC precursors may be evaluated based on factors including, but not limited to: (1) recovery and viability after cryopreservation and thawing; and (2) the ability to differentiate ex vivo into mature DC.
  • cryopreservation on DC products may be evaluated based on factors including, but not limited to: (1) recovery and viability after cryopreservation and thawing; and (2) changes in phenotype and function due to cryopreservation.
  • the examples provided herein include specific reference to a particular concentration of HSA, it will be understood that the amount of HSA in the freezing medium may vary, but is generally in the range of 1% to 30% (v/v or volume percent) . However, any concentration of HSA that results in a cell viability of at least 50% and a cell recovery of at least 50%, and preferably a cell viability and recovery of at least 70 or 80% may be used in the DC precursor and Ag-loaded DC compositions, as well as the cryopreservation methods described herein.
  • DMSO concentrations of from about 5% to as high as 20% (v/v or volume percent) may be included in the cryopreservation methods described herein.
  • concentrations of DMSO e.g., about 5% to 10%, however, any concentration of DMSO that results in a cell viability of at least 50% and a cell recovery of at least 50%, and preferably a cell viability and recovery of at least 70 or 80% may be used in the DC precursor and Ag-loaded DC compositions, as well as the cryopreservation methods described herein.
  • a rate-controlled freezing system Forma
  • numerous methods of freezing in a rate-controlled or non-rate-controlled manner are routinely employed by those of skill in the art.
  • various serum-free media are commercially available and may be used in carrying out the invention. Examples include XVIVO 10, XVIVO 15, XVIVO 20, StemPro and any commercially available serum- free media.
  • cryopreservation examples refer to 10% DMSO
  • DMSO concentrations of from about 5% to as high as 20% may be included in the cryopreservation methods described herein.
  • lower concentrations of DMSO are preferred, e . g. , about 5% to 10%.
  • any freezing procedure, type of serum-free medium and concentration of DMSO that results in a cell viability of at least 50% and a cell recovery of at least 50%, and preferably a cell viability and recovery of at least 70 or 80% may be used in the DC precursor and Ag-loaded DC compositions, as well as the cryopreservation methods described herein .
  • Cryopreserved DC precursors were cultured ex vivo under conditions effective to generate mature DC and the percent (%) recovery and % viability of the cells compared to DC generated from fresh precursors, as described in Example 2.
  • Mature DC were also generated ex vivo from fresh DC precursors in the presence or absence of tumor Ag. They were then cryopreserved on either a small or large scale under the same conditions as DC precursors, as described in Example 2.
  • DC precursors differentiate into mature, competent DC with allostimulatory and Ag-presenting abilities.
  • DC generated from both fresh and cryopreserved DC precursors were allostimulatory, with no significant difference in the observed allostimulatory activity between DC derived from fresh versus cryopreserved precursors.
  • DC products generated from either cryopreserved precursors or cryopreserved as mature DC products.
  • DC products generated from either cryopreserved precursors (Table VA) or cryopreserved as DC products (Table VB) , were stable in lactated Ringer's solution, with a high percentage recovery and viability for up to 24 hours.
  • the stability of DC products was also demonstrated in phenotypic analysis and functional assays, which indicate that DC products, generated from either cryopreserved precursor (Table VIA, Fig. 4A) or cryopreserved as mature DC products (Table VIB, Fig. 4B) , maintain their phenotype and allostimulatory activity for up to 24 hours at 4°C in lactated Ringer's solution.
  • DC precursors enriched from peripheral blood may be cryopreserved on both a small and large scale in liquid nitrogen for a period of 2-24 weeks in serum-free cryomedia.
  • the duration of storage can be extended indefinitely beyond 24 weeks without impacting cell recovery, viability, phenotype or function.
  • Good recovery and viability is observed upon thawing the cells and the cryopreservation conditions described herein do not affect the ability of DC precursors to differentiate into mature DC, as evidenced by both phenotype and function.
  • cryopreserved DC products The recovery and viability of cryopreserved DC products is comparable to that of cryopreserved precursors, and cryopreservation of DC products did not affect their phenotype or function.
  • cryopreserved DC precursors include but are not limited to, one or more of:(l) cellular immunotherapy with a uniform cell population which may be administered at multiple time points; (2) utility as a base line sample for clinical monitoring following DC immunotherapy and other vaccination studies; (3) utility as a diagnostic and prognostic tool; (4) utility in ex vivo and in vivo generation of T cells specific to naive or weak Ag; (5) utility in generation of immune chimeras for allo- and xeno- transplantation; (6) utility in gene therapy; (7) utility in various research and development activities; and (8) utility in immunomodulatory therapies using autologous, allogeneic or xenogeneic DC for both stimulating Ag-specific immune responses and treating autoimmune disease.
  • Cells that have been cryopreserved under serum-free conditions provide the advantage of being free of infectious agents, foreign proteins (which may be antigenic) , antibodies, etc., that are typically found in serum. Accordingly, cryopreserved under serum-free conditions can be directly reinfused into patients after thawing. Additionally, serum-free conditions are more reproducible without the lot-to-lot variability that is inherent in any serum preparation, assuring better quality control of products prepared under such conditions .
  • PBMC peripheral blood mononuclear cells
  • DC precursors were isolated by either a one-step or a successive two-step buoyant density centrifugation procedure using buoyant density solutions, BDS 77 and BDS 65 (Dendreon Corp.).
  • Autologous T cells were purified from leukapheresis products by either a one-step buoyant density centrifugation procedure using BDS 77, or two-step buoyant density centrifugation using BDS 77 and BDS 65, followed by negative selection affinity chromatography (R&S Systems) . Allogeneic T cells were enriched from buffy coat preparations by the same method as autologous T cells.
  • mAb Monoclonal antibodies
  • Commercially available mAb were used to stain DC and DC precursors for flow cytometry including the following: (1) the fluorescein (FITC) conjugated antibodies, CDla (Biosource, BS), CD14 (Becton Dickinson, BD) , CD66b (Coulter/Immunotech, Cl), HLA-DR (BD) , Lin 1 (BD) and IgGl (BD) ; (2) the PE-conjugated antibodies, CD3 (BD) , CDllc (BD) , CD19 (BD), CD40 (BD) , CD54 (BD) , CD56 (BD) , CD86 (Pharmingen) and IgGl (BD) ; and (3) the PerCP-conjugated antibodies HLA-DR (BD) and IgG2a (BD) .
  • FITC fluorescein
  • CDla Biosource, BS
  • CD14 Becton Dickinson, BD
  • CD66b Coulter/I
  • DC precursors were cultured in Teflon bags (American Fluoroseal) at a density of lxl0 7 /ml in Aim-V medium supplemented with 2 mM glutamine in a humidified incubator at 37°C under 5% C0 2 for 40 hours. During the culture period DC precursors were pulsed with Ag, PA2024 at 10 ⁇ g/ml or HER500 at 20 ⁇ g/ml.
  • MLR Mixed Lymphocyte Reaction
  • Ag presentation assays DC precursors were cultured ex vivo for 40 hours in the presence or absence of Ag .
  • Exemplary antigens include PA2024 and HER500, as further described below.
  • Ag-loaded DC were then washed and used for assays either immediately following culture or following cryopreservation and thawing.
  • the T cell stimulatory activity of the Ag-loaded DC was measured by incubating 800 - 8 xlO 5 Ag-loaded DC with 1x10" autologous T cells per well in triplicate wells of 96-well round bottom plates. Ag was not added during the assay period. Proliferation of T cells was measured as above.
  • Antigens (Ag) .
  • Two exemplary recombinant tumor Ag evaluated herein were: (1) recombinant PA2024, a fusion protein consisting of PAP and human granulocyte macrophage-colony stimulating factor (hGM-CSF) ; and (2) recombinant HER500, a fusion protein comprising 300 amino acids of the N-terminal extracellular domain and 200 amino acids of the C-terminal intracellular domain of HER-2/neu and hGM-CSF.
  • hGM-CSF human granulocyte macrophage-colony stimulating factor
  • Cryopreserved DC precursors were cultured ex vivo in AIM-V serum-free medium in the presence of PA2024. After 40 hours of culture, cells were harvested and their recovery and viability examined as above. The recovery of DC generated from cryopreserved precursors in the presence of PA2024 was 80.4 ⁇
  • DC products were generated ex vivo from fresh DC precursors in the presence or absence of tumor Ag . They were then cryopreserved on either a small or large scale under the same conditions as DC precursors. After 4-16 weeks of cryopreservation, DC products were thawed and their recovery and viability examined. As shown m Table IIIA, the mean recovery of PA2024-loaded DC cryopreserved on a small scale was 69.1 ⁇ 16.0% with a viability of 80.0 ⁇ 9.6%, while that of DC products cryopreserved on a large scale was 87.7 ⁇ 2.4% with 98.2 ⁇ 2.6% viability (Table IIIB) .
  • cryopreservation The effect of cryopreservation on the ability of DC precursors to mature was assessed by comparing the cell surface expression of molecules typically expressed on mature DC following ex vivo culture for 40 hours, as described above.
  • the FACS profile of DC generated from fresh precursors and those generated from cryopreserved precursors was compared by gating on the Lin-negative/HLA-DR-positive population with analysis focused on evaluating the expression of CDllc, CD40, CD54 and CD86 on DC lineage cells.
  • the effect of cryopreservation on DC cryopreserved at the stage of mature Ag- loaded DC was also examined.
  • DC precursors differentiate into mature, competent DC with allostimulatory and Ag-presenting abilities.
  • the Ag-presenting ability of DC generated from fresh precursors versus DC derived from cryopreserved precursors was compared in a MLR.
  • Ag-specific T cell stimulatory activity of the DC was also examined in T cell proliferation assays using autologous T cells and Ag-loaded DC generated in the presence of the recombinant tumor Ag, PA2024 or HER500.
  • DC generated from both fresh and cryopreserved precursors were allostimulatory as shown in Fig. 2A. There was no significant difference in the observed allostimulatory activity between DC derived from fresh and cryopreserved precursors.
  • cryopreservation does not affect the ability of DC precursors to differentiate into mature, competent DC, that are not only allostimulatory but also capable of stimulating autologous T cells in an Ag-specific manner.
  • cryopreservation of DC products does not affect their allostimulatory and Ag-presenting abilities.
  • DC products cryopreserved at the stage of mature Ag-loaded DC were as stimulatorynm as fresh DC to allogeneic (Fig. 2A) and autologous (Fig. 2B-C) T cells.
  • DC products generated either from cryopreserved precursors or DC cryopreserved as mature DC products were assessed using the small scale cryopreservation methods described herein.
  • DC products generated from cryopreserved precursors were resuspended immediately after their harvest at lxl0 7 /ml in lactated Ringer's solution, while DC cryopreserved as DC products were thawed and resuspended as above.
  • DC products generated either from cryopreserved precursors or cryopreserved as DC products, were stable in lactated Ringer's solution up to 24 hours with a recovery of 88.7 ⁇ 11.8% and a viability of 87.1 ⁇ 4.7% (Table VA)
  • DC cryopreserved as DC products had a recovery of 86.9 ⁇ 22.1% and a viability of 75.4 ⁇ 17.2% (Table VB) .
  • DC products either generated from cryopreserved precursors (Fig. 4A) or cryopreserved as mature DC products (Fig. 4B) , maintained their allostimulatory activity for up to 24 hours at 4°C in lactated Ringer's solution. Together, these results indicate that DC products, either generated from cryopreserved precursors or cryopreserved as DC products, are stable under the above conditions for up to 24 hours.

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Abstract

Cette invention concerne un précurseur de cellule dendritique soumise à une cryoconservation sans sérum et une composition de cellules dendritiques chargées d'antigènes ainsi qu'une technique permettant d'obtenir une telle composition. Cette technique consiste conserver à très basse température des cellules d'une manière efficace en termes de récupération, viabilité, maintien de l'aptitude à se différencier en cellules dendritiques matures pour des précurseurs de telles cellules et maintien du phénotype de la cellule dendritique et de la fonction antigénique spécifique pour des cellules dendritiques matures chargées d'antigènes.
PCT/US2000/032722 1999-12-03 2000-12-01 Cryoconservation of de cellules dendritiques chargees d'antigenes et leurs precurseurs dans des milieux sans serum WO2001039594A2 (fr)

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